Many GTPases that participate in signal transduction, including those that regulate inflammatory and neoplastic cells, are among a large family of proteins that are targeted to membranes by a series of posttranslational modifications of a C-terminal CAAX motif that includes prenylation, proteolysis, and carboxyl methylation. Of these, only carboxyl methylation is reversible under physiologic conditions and may therefore serve a regulatory function. Because mutations of the CAAX motif that block processing eliminate all three sequential modifications, the specific role of carboxyl methylation can only be determined by disrupting the methyltransferase. Mammalian prenylcysteine carboxyl methyltransferase (pcCMT) had defied molecular characterization because the active enzyme could not be extracted from membranes and because homology cloning from the yeast gene failed. Using an EST fragment, we have recently cloned and sequenced mammalian pcCMT from a human myeloid cell line (HL60). Preliminary studies using GFP fusion proteins revealed that pcCMT is expressed in ER and Golgi but excluded from the plasma membrane, suggesting that CAAX proteins such as ras that are targeted to the plasma membrane in a methylated form must first transit internal membranes. In this proposal we will utilize the pcCMT gene to study the biological role of prenylcysteine carboxyl methylation. Specific Aim 1: Biological function of pcCMT in vitro. We will overexpress by transfection or underexpress using antisense oligonucleotides, antisense plasmids, or pharmacologic inhibitors, pcCMT and assess the function of ras, rho family GTPases, and G proteins using a variety of well-established assays. Specific Aim 2: Biological function of pcCMT in vivo: construction of a pcCMT null mouse. We will isolate a genomic clone of murine pcCMT and construct, by targeted gene disruption, a pcCMT null mouse. If viable, we will characterize any overt phenotype and study GTPase function in vivo, in leukocytes, and in cell lines derived form the null mouse. Specific Aim 3: Structure/function analysis of the human myeloid pcCMT gene product. Employing assays of in vitro enzymatic activity, ste14 complementation, GFP subcellular localization, and any biologic readouts identified in Specific Aim 1. we will conduct a structure/function analysis of the pcCMT gene product by systematic mutational analysis. Specific Aim 4. Membrane targeting of pcCMT substrates: relationship between CAAX processing and vesicular transport. Using pulse-chase/subcellular fractionation, GFP labeling, and immunogold electron microscopy, we will test our hypothesis that CAAX proteins are targeted to the plasma membrane via the cytoplasmic side of the secretory apparatus and that carboxyl methylation is required for this process. Elucidation of the biological role of pcCMT together with structure/function analysis will facilitate targeting this enzyme for the development of inhibitors with potential as novel antiinflammatory and/or anticancer drugs.